Actin is an established, key component of the neuronal cytoskeleton, involved in axon elongation, signaling, and synaptic homeostasis. While actin organization and dynamics have been well-studied in terminal growth- cones, even the basic organization and dynamics of actin along axon-shafts is unclear. One reason for this gap in knowledge has been paucity of tools for live imaging of filamentous (F-actin). Like most cytoskeletal proteins, actin exists as both freely diffusible monomers and polymers, and labeling the entire actin pool invariably creates a diffusive background that makes imaging difficult. Fortunately, recently-developed actin probes that selectively bind to F-actin circumvent this issue. Using such probes in preliminary studies, my colleagues and I found focal actin 'hotspots' distributed along axons; where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin assembly, generating long intra-axonal filaments (actin trails) that seem to the basis for axonal architecture. Notably, F-actin hotspots co-localize with stationary axonal endosomes, suggesting nucleation on their surface. Our studies also suggest that these actin dynamics are dependent on formins, a family of actin nucleators/elongators, known to be mutated in some forms of Autism Spectrum Disorders (ASD). Collaborative super-resolution studies further solidify these observations. These preliminary studies have given rise to a new model where axonal actin dynamically localizes on the surface of vesicles to assist in axonal growth and synaptic homeostasis. My overall goal is to test the cell-biological predictions of this model. Specifically, my aims are: (1) to determine the organization and polarity of intra-axonal actin filaments, fundamental issues that are currently unknown, (2) to determine the mechanisms generating intra-axonal actin filaments, and (3) to determine mechanisms severing intra-axonal actin filaments. Working towards this thesis will not only help resolve the huge gap in knowledge in the neuronal cytoskeleton field, but also lay the foundation for understanding putative abnormalities of actin dynamics in ASD. Besides serving as a platform to get trained in doing rigorous science, I believe my endeavors will also help towards my goal of being a pediatric neurologist doing biomedical research.